Tumor hypoxia has been shown to decrease tumor control and survival in head and neck (H&N) cancer patients treated with radiotherapy. With the development of novel and effective therapies against hypoxia, there is a need for a simple and non-invasive way to identify patients who would benefit from such treatments. Presently, the most widely accepted method for detecting tumor hypoxia is the Eppendorf pO2 histograph, which is an invasive and cumbersome technique that requires high level of patient cooperation. In addition, it can not be used for deeply seeded tumors. The comet assay, which assesses single stranded DNA breaks of individual tumor cells after a large radiation dose, is another approach used to measure tumor hypoxia. Similar to the polargraphic method, it is an invasive procedure, whose success depends on tumor accessibility and expertise. We have had extensive experience using both methods to detect tumor hypoxia in H&N cancer patients. A powerful approach for the discovery of unique genes is by microarray technology, which can be used to study global gene expression changes by tumor hypoxia. Many hypoxia-induced genes are crucial for tumor growth and dissemination and code for secreted proteins that can be measured in serum. We hypothesize that DNA microarray analysis of membrane-associated polysomes can be used for large-scale identification of hypoxia-induced genes with secreted proteins. We propose to perform microarray studies in 4 H&N cancer cell lines exposed to 2 different time courses of hypoxia. We will use a noel technique for microarray statistical analysis to identify candidate hypoxia-induced genes that code for secreted products. We will determine the full-length cDNA sequence of induced ESTs. We will generate antisera to novel peptides, which will be used to determine the sub-cellular location of these proteins in vitro. We will use ELISA to measure the serum level of the secreted proteins in SCID mice harboring transplanted human tumor made hypoxic by bleeding, to identify candidate hypoxia-induced secreted proteins that are measurable in the blood. We will also employ immunohistochemical staining of tumor explains to establish in-vivo localization of these proteins and to correlate their expression with EF5 staining. Finally, we will use ELISA to measure in patients' serum those proteins that are found to be measurable in mouse blood. In this group of H&N cancer patients, we will also measure tumor pO2 and tumor hypoxic fraction with the Eppendorf pO2 histograph and the comet assay. Comparison of the 3 methods will determine the feasibility of the newly developed panel of blood tests in identifying tumor hypoxia.